Chlorite in the Treatment of Neurodegenerative Disease

ABSTRACT

The invention features methods of treating a macrophage-associated neurodegenerative disease such as amyotrophic lateral sclerosis (ALS), Alzheimer&#39;s disease (AD), or multiple sclerosis (MS) in a subject by administering chlorite in an amount effective to decrease blood immune cell activation. The invention also features methods of monitoring therapy by assessing blood immune cell activation before and after therapy.

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application Ser.No. 60/541,576, filed Feb. 3, 2004, which application is incorporatedherein by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with government support under federal grant no.U01-CA66529 awarded by the National Institutes of Health. The UnitedStates Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention generally relates to the use of chlorite intreatment of neurodegenerative disease, particularly a neurodegenerativedisease characterized by pathologic macrophages, such as amyotrophiclateral sclerosis (ALS), multiple sclerosis (MS), HIV-associatedneurological disorders, or Alzheimer's disease (AD).

BACKGROUND OF THE INVENTION

Neurodegenerative diseases are generally characterized by a degenerationof neurons in either the brain or the nervous system of an individual.Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), andmultiple sclerosis (MS) fall within this category. These diseases aredebilitating, the damage that they cause is often irreversible, and theoutcome in a number of cases is fatal.

ALS is characterized by gradual degeneration of motor neuron cells inthe spinal cord and brain, which ultimately leads to progressiveweakness and paralysis of muscle and death. ALS occurs in two clinicallyindistinguishable forms, referred to as a sporadic form and a familialform. The pathogenesis of ALS is incompletely understood, althoughdifferent hypotheses have been suggested, including mitochondriadysfunction, mutation in the superoxide dismutase gene, and defects inneuronal glutamate transport. Autoimmunity has also been hypothesized tobe involved in ALS pathogenesis (Appel et al. 1993. J. Neurol. Sci.118:169-174). In addition, several recent studies have suggested thatthe immune system may be actively involved in the disease process ofALS, with observations of activated microglia, IgG deposits, increasedFcR expression, and dysregulation of cytokine expression in the spinalcord of ALS patients (Troost et al. 1989. Clin. Neuropathol. 8:289-294;Engelthardt et al. 1990. Arch. Neurol. 47:1210-1216; Schiffer et al.1996. J. Neurol. Sci. 139(suppl):27-33; Hayashi et al. 2001 J. Neurol.Sci. 188:3-7.9-12).

Recent clinical and pathological studies have shown that involvementoutside the motor neuron system is relatively common in AmyotrophicLateral Sclerosis (ALS) (Hayashi et al. 2001, supra; Obal et al. 2001Neuroreport. 12:2449-2452; Sola et al. 2002 8. Eur. Neurol. 47:108-112;Ono et al. 2001 J. Neurol. Sci. 187:27-34; Alexianu et al. 2001Neurology. 57:1282-1289.). Microglia/macrophage activation andinflammatory response have been implicated in ALS disease progression(Appel et al. 1993, supra; Engelthardt et al. 1990, supra; Hayashi etal. 2001 supra; Obal et al. 2001 Neuroreport. 12:2449-2452; McGeer etal. 2002 Muscle Nerve. 26:459-47028, 29). However, few studies to datehave explored the status of the systemic immune response in ALS. Despiteintensive investigation, ALS has no known cause or effective therapy.

Retroviral infection has recently been implicated in the pathogenesis ofan ALS-like syndrome in patients with HIV-associated disease. Moulignieret al. (Reversible ALS-like disorder in HIV infection. Neurology.57:995-1001) recently reported the outcome of six HIV-1-infectedpatients with a neurologic disorder mimicking ALS and all those patientsstabilized or improved with antiretroviral therapy. MacGrowen et al.(2001. An ALS-like syndrome with new HIV infection and complete responseto antiretroviral therapy. Neurology. 57:1094-10) also reported adramatic clinical response to antiretroviral therapy in an ALS-likesyndrome with new HIV infection.

Approximately one-quarter of individuals with AIDS developneuropathological symptoms. Infection by HIV-1 causes inflammationwithin the brain and neuronal degeneration (Power et al. 2001 Adv.Virus. Res. 56:389-433), resulting in HIV-associated dementia (HAD) orthe less severe minor cognitive and motor disorders (Janssen et al. 1991Report of a Working Group of the American Academy of Neurology AIDS TaskForce. Neurology. 41:778-785; McArthur et al. 1993 Multicenter AIDSCohort Study. Neurology. 43:2245-2252; The Dana Consortium. 1996.Clinical confirmation of the American Academy of Neurology algorithm forHIV-1-associated cognitive/motor disorder. The Dana Consortium onTherapy for HIV Dementia and Related Cognitive Disorders. Neurology.47:1247-1253.).

The mechanisms underlying HIV-associated neuronal injury areincompletely understood. Various studies have suggested thatmonocytes/macrophage activation may play a significant role in thepathogenesis of many neurological diseases (Smits et al. 2000 Eur. J.Clin. Invest. 30:526-535; Fiala et al. 2002 Eur. J. Clin. Invest.32:360-371; Minagar et al. 2002 The role of macrophage/microglia andastrocytes in the pathogenesis of three neurologic disorders:HIV-associated dementia, Alzheimer disease, and multiple sclerosis. J.Neurol. Sci. 202:13-23), including HIV-associated neurologic disorders(Pulliam et al. 1997 Lancet. 349:692-695; Diesing et al. 2002 AIDSReader. 12:358-368). Indeed, the best pathological correlate forHIV-associated neurologic disorders, especially HAD, is the number ofactivated mononuclear phagocytes (perivascular and parenchymalblood-derived macrophages and microglia), not the absolute levels ofvirus in brain per se (Glass et al. 1995 Ann. Neurol. 38:755-762;Adamson et al. 1999 Mol. Med. 5:98-109). Similar findings have beenreported for simian AIDS related encephalopathy (SIVE) (Williams et al.2002 Am. J. Pathol. 161:575-585). Macrophage activation has beenreported in spinal cords of patients with ALS disease (Appel et al.1993, supra; Engelthardt et al. 1990, supra; Obal et al. 2001, supra;McGeer et al. 2002, supra), although the role of macrophage activationin ALS pathogenesis has not been previously determined.

Studies on blood from patients with HAD (Liu et al. 2000 J. Neurovirol.6(suppl 1): S70-81) and monkeys with SIVE (Williams et al. 2002 Am. J.Pathol. 161:575-585) have shown a direct relationship between thepresence of activated blood macrophages and central nervous system (CNS)disease. These activated macrophages are thought to mediate blood brainharrier (BBB) breakdown and directly contribute to CNS pathogenesis.

Alzheimer's disease (AD) is the most common form of dementia among theelderly. Various studies have suggested that macrophage activation maybe involved in AD (see, e.g., WO 99/21542). Currently the only definiteway to diagnose AD is by post-mortem autopsy to assess the presence ofamyloid plaques and tangles in brain tissue. Thus, AD diagnosis isgenerally a diagnosis of “possible” or “probable” AD. At specializedcenters, doctors can diagnose AD correctly up to 90 percent of the time.Several tools are used to diagnose “probable” AD, including medicalhistory, analysis of blood urine, or spinal fluid, to rule out othercauses (e.g., thyroid deficiencies, infectious disease, etc.), brainscans, and neuropsychological tests to asses memory, problem solving,attention, counting, and language.

Multiple sclerosis (MS) is a chronic disease that is characterized by“attacks,” during which areas of white matter of the central nervoussystem, known as plaques, become inflamed. Inflammation of these areasof plaque is followed by destruction of myelin, the fatty substance thatforms a sheath or covering that insulates nerve cell fibers in the brainand spinal cord. Myelin facilitates the smooth, high-speed transmissionof electrochemical messages between the brain, spinal cord, and the restof the body. Damage to the myelin sheath can slow or completely blockthe transmission of these electrochemical messages, which can result indiminished or lost bodily function.

The most common course of MS manifests itself as a series of attacks,which are followed by either complete or partial remission, during whichthe symptoms lessen only to return at some later point in time. Thistype of MS is commonly referred to as “relapsing-remitting MS.” Anotherform of MS, called “primary-progressive MS,” is characterized by agradual decline into the disease state, with no distinct remissions andonly temporary plateaus or minor relief from the symptoms. A third formof MS, known as “secondary-progressive MS,” starts as arelapsing-remitting course, but later deteriorates into aprimary-progressive course of MS.

The symptoms of MS can be mild or severe, acute or of a long duration,and may appear in various combinations. These symptoms can includevision problems such as blurred or double vision, red-green colordistortion, or even blindness in one eye, muscle weakness in theextremities, coordination and balance problems, muscle spasticity,muscle fatigue, paresthesias, fleeting abnormal sensory feelings such asnumbness, prickling, or “pins and needles” sensations, and in the worstcases, partial or complete paralysis. About half of the people sufferingfrom MS also experience cognitive impairments, such as for example, poorconcentration, attention, memory and/or judgment. These cognitivesymptoms occur when lesions develop in those areas of the brain that areresponsible for information processing.

Despite the progress in the field, there remains a need for a therapyfor treatment of ALS and MS, including alleviation of symptoms of thesediseases. The present invention addresses this need.

LITERATURE

The following references, as well as those in the Bibliography and citedthroughout, may be of interest: Akiyama et al. 2000 Neurobiol. Aging.21:383-421; Anderson et al. 2002. J. Leukoc. Biol. 72:101-106; Cremer etal. 1976. N. Engl. J. Med. 295:107-108. (Letter); Fischer-Smith et al.2001. J. Neurovirol. 7:528-541; Giese et al. Cell Immunol. 2004 June;229(2):149-58; Hansen et al. Pharmacol Toxicol. 2001 August; 89(2):92-5;Hensley et al. 2002. J. Neurochem. 82:365-374; Hirsch et al. 2003. Ann.N.Y. Acad. Sci. 991:214-228; Kemp et al. Pharmacol Toxicol. 2002 June;90(6):346-8; Kemp et al. Transplant Proc. 2000 August; 32(5):1018-9;Klaustermeyer et al. 1989. Ann. Allergy. 63:327-330; Kott et al. 1979.Neurology. 29:1040-1044; Lehrich et al. 1974. J. Neurol. Sci.23:537-540; Marshall et al. 1998. Brain Behav. Immun. 12:297-307; McGeeret al. 1998. Exp. Gerontol. 33:371-378; Morgan et al. 1988. Arch. Dis.Child. 63:771-773; Nottet et al. 1996. J. Immunol. 156:1284-1295;Provinciali et al. 1988. Acta. Neurol. Scand. 78:449-454; Nguyen et al.2001 Ann. Neurol. 50:630-639; Ostermeyer-Shoaib et al. 1993 Acta NeurolScand. 87:192-194; Raffanti et al. Infection. 1998 July-August;26(4):202-7; Schempp et al. Arzneimittelforschung. 2001; 51(7):554-62;Tikka et al. 2001. J. Neurosci. 21:2580-2588; Veerasarn et al. RadiotherOncol. 2004 November; 73(2):179-85. and Van Den Bosch et al. 2002.Neuroreport. 13:1067-1070.

See also: McGrath et al. 2002 Curr. Opin. Investig. Drugs 3:365-373;McGrath et al. 1999 Pathobiology. 67:277-81; McGrath et al. 1998Transplant. Proc. 30:4200-4204; Giese et al. 2004 June; 229(2):149-58;Zhang et al. J Neuroimmunol. 2005 February; 159(1-2):215-24. Epub 2004Nov. 26.

Also see: U.S. Pat. Nos. 4,725,437; 5,877,222; 6,086,922; and U.S.Publication Nos. 20030175832; 20030158262; 20030130357; and 20030130350.

SUMMARY OF THE INVENTION

The invention features methods of treating a macrophage-associatedneurodegenerative disease such as amyotrophic lateral sclerosis (ALS),Alzheimer's disease (AD), or multiple sclerosis (MS) in a subject byadministering chlorite in an amount effective to decrease blood immunecell activation. The invention also features methods of monitoringtherapy by assessing blood immune cell activation before and aftertherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. Included in thedrawings are the following figures:

FIG. 1 is a graph showing the relationship of the revised ALS FunctionalRating Score (ALSFRS-R) to CD4 T-cell co-expression of the activationantigen CD38 in ALS patients. Patients with ALS were divided into twogroups based on a score of 24, the midpoint of the ALSFRS-R scale. TheCD4 activation marker CD38 was significantly lower in patients withsevere impairment (ALSFRS-R score of 0-24, n=10) compared to normalcontrols (P<0.01) and patients with milder impairment (ALSFRS-Rscore >24, n=26) (P<0.05), but no difference was found between normalcontrols and patients with milder impairment.

FIGS. 2 a-2 b are graphs showing analyses of macrophage activationdefined by CD14 co-expression of HLA-DR in patients with ALS. FIG. 2 ais a graph showing a negative correlation of macrophage activation withALSFRS-R scores in patients with ALS (Pearson r=−0.3424, P=0.0409). FIG.2 b is a graph showing a positive correlation of levels of HLA-DR on ALSCD14 cells with the rate of disease progression (ALSFRS-R score changeper month) (Pearson r=0.3696, P.=0.0265).

FIGS. 3 a-3 b are graphs showing a comparison of serum-IgG and -IgMlevels between normal controls and ALS patient groups by ALSFRS-Rcategories. FIG. 3 a is a graph showing that significantly lower levelsof serum-IgG were found in ALS patients with severe impairment comparedto normal controls (P<0.05), but with no difference between patientswith milder impairment and normal controls. FIG. 3 b is a graph showinglevels of serum-IgM in patients with milder impairment weresignificantly higher than normal controls (P<0.01), but with nodifference between patients with severe impairment and normal controls.

FIG. 4 is a graph showing the effects of WF10 administration uponactivated blood macrophages in an ALS patient.

FIG. 5 is a graph of a composite set of curves representing bloodmacrophage activation measurements taken from a patient with multiplesclerosis treated one cycle of WF10.

FIG. 6 is a graph showing the results of administration of WF10 to twoALS patients (WF10, 1 cycle=0.3 ml WF10/kg infused over 1 hr, withinfusion daily for 5 days, which 5-day regimen was administered onceevery 3 weeks; data shown at the end of 4 cycles and at the end of 5cycles. The arrows indicate the periods during which a WF10 cycle wasadministered. Results are indicated in terms of an ALS/FR (amyotrophiclateral sclerosis/functional) score. G-tube indicates period duringwhich gastric tube was in place. Oral indicates the patient was able totake food by mouth.

Before the present invention is described in more detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “achlorite matrix” includes a plurality of such chlorite matrices andreference to “the composition” includes reference to one or morecompositions and equivalents thereof known to those skilled in the art,and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION OF THE INVENTION Overview

The invention is based on the discovery that administration of WF10,which comprises chlorite (e.g., in the form of tetrachlorodecaoxygen(TCDO)) as its active ingredient, provides for treatment of patientshaving amyotrophic lateral sclerosis (ALS) and for treatment of patientshaving multiple sclerosis (MS). Without being held to theory, chloriteprovides for a decrease in activated blood immune cells (e.g., activatedmacrophages), which are elevated in ALS and MS patients and contributeto ALS and MS disease pathogenesis. Further, since Alzheimer's disease(AD) is similarly characterized as being associated with activated bloodimmune cells in a manner that parallels ALS, AD is also amenable totreatment by administration of WF10. The invention is thus applicable totreatment of neurodegenerative diseases associated with activated bloodimmune cells, particularly with proliferating or inappropriate activatedmacrophages.

DEFINITIONS

A “neurodegenerative disease” refers to a central nervous systemcharacterized by progressive, normally gradual, loss of functionalneural tissue. Of particular interest in the present invention is thetreatment of neurodegenerative diseases in which that affected patienthas activated blood immune cells, particularly with proliferating orinappropriate activated macrophages. Reference to a “non-diseased”individual generally means an individual who is not diagnosed as having,or is not suspected of having, the relevant neurodegenerative disease.Reference to a “diseased” individual generally means an individual whohas been diagnosed as having, or who is suspected of having, therelevant neurodegenerative disease. Exemplary neurodegenerative diseasesinclude amyotrophic lateral sclerosis, multiple sclerosis, andpathogen-mediated or pathogen-associated neural diseases or symptoms(such as viral infection, e.g., HIV infection).

As used herein, the terms “macrophage” and “monocyte” are usedinterchangeably, as it is understood that in the art the term “monocyte”is often used to describe a circulating mononuclear cell that expressesthe CD14 cell surface marker, and when in a tissue this cell is alsoclassified as a macrophage.

An “abnormal macrophage” or “activated circulating monocyte” or“activated monocyte” as used interchangeably herein denotes a monocytewhich expresses CD14 (i.e., CD14+) and which expresses an elevated levelof HLA-DR, the major histocompatibility antigen class II, and/or whichexpresses CD16 (i.e., CD16+). Generally, abnormal macrophages are foundin peripheral blood but they may also be found in other biologicalsamples from an individual. Generally, these abnormal macrophages arepresent without identifiable concomitant T cell activation in the ALSpatients.

As used herein, detecting the “presence of abnormal macrophages”generally means detecting the level of abnormal macrophages. Generally,the level of abnormal macrophages (or activated monocytes) is indicatedby the level of HLA-DR expression in a population of CD14+ cells and/orthe percentage of CD16+ cells in a population of CD14+ cells and/or thenumber of CD14+/CD16+ cells, although other markers that indicatemonocyte activation, differentiation and/or proliferation could be used.It is understood that an absolute or even relative level need not bedetermined; an observation of detectable abnormal macrophages issufficient.

A “proliferating macrophage” or “promac” is understood in the art and asused herein denotes a disease-associated blood macrophage which exhibitsan elevation in proliferation and/or activation markers relative tonon-disease blood macrophages. Normally a macrophage is a terminallydifferentiated cell incapable of further division. For purposes of thisinvention, a “proliferating macrophage” is capable of further divisionor is in a portion of the cell cycle not considered to be terminal orend stage, and/or has undergone inappropriate activation (e.g., are“inappropriately activated”) or is undergoing inappropriate activation.Methods of detecting proliferating macrophage(s) are discussed below.

“Pathologic macrophages” as used herein is meant to encompass bothproliferating macrophages and inappropriately activated macrophages(e.g., abnormal macrophages). Pathologic macrophages thus encompassproliferating macrophages, as defined above, as well as macrophages inthe blood that may not exhibit proliferation markers at any given time,but are nonetheless chronically activated, and thus are in a pathogenicstate.

As used herein, detecting the “presence of proliferating macrophages”generally means detecting the level of proliferating macrophages. It isunderstood that an absolute or even relative level need not bedetermined; an observation of detectable proliferating macrophages issufficient.

A “macrophage-associated” disease, disorder or indication is a disease,disorder or indication that is associated with pathologic macrophages anelevated, or abnormal, level or rate of macrophage proliferation ascompared to control sample(s). Such disorders include, but are notlimited to, macrophage-associated neurodegenerative disorders, such asALS, MS, HIV-associated neurological disorders, and AD. The terms“disorder” and “disease” are used interchangeably herein. An“HIV-associated” disease is defined more broadly as generally associatedwith or secondary to an HIV infection; “HIV-mediated” diseases, forexample, are included in those considered to be “HIV-associated.” Inparticular embodiments, the disorder contemplated for treatmentaccording to the invention is not cancer (e.g., is a disease or disorderother than cancer). In other particular embodiments, the disordercontemplated for treatment according to the invention is not anautoimmune disease (e.g., the macrophage-associated is a disease ordisorder other than an autoimmune disorder or disease). For example, thedisorder is not graft rejection (transplant rejection). In otherembodiments, the disorder treated is a viral infection, particularly anHIV or HCV infection (i.e., the patient is not virally infected, e.g.,is not HIV-infected or HCV-infected) In still other embodiments, thedisorder is not have HIV-associated dementia (e.g., the patient does nothave AIDS dementia), “Macrophage-associated neurodegenerative disorder”is specifically defined herein to exclude cancer, HIV infection, HCVinfection, and autoimmune diseases.

A “macrophage-associated neurodegenerative disorder” is aneurodegenerative disease in which the patient has pathologicmacrophages (e.g., abnormally activated macrophages and/or proliferatingmacrophages, particularly a disease associated with an elevated, orabnormal, level or rate of macrophage proliferation as compared tocontrol sample(s)). “Macrophage-associated neurodegenerative disorder”is specifically defined herein to exclude cancer and autoimmunediseases.

“Amyotrophic lateral sclerosis” or “ALS” are terms understood in the artand as used herein to denote a progressive neurodegenerative diseasethat affects upper motor neurons (motor neurons in the brain) and/orlower motor neurons (motor neurons in the spinal cord) and results inmotor neuron death. As used herein, the term “ALS” includes all of theclassifications of ALS known in the art, including, but not limited toclassical ALS (typically affecting both lower and upper motor neurons),Primary Lateral Sclerosis (PLS, typically affecting only the upper motorneurons), Progressive Bulbar Palsy (PBP or Bulbar Onset, a version ofALS that typically begins with difficulties swallowing, chewing andspeaking), Progressive Muscular Atrophy (PMA, typically affecting onlythe lower motor neurons) and familial ALS (a genetic version of ALS).

“Multiple sclerosis” or “MS” are terms understood in the art and as usedherein to denote a progressive neurodegenerative disease resulting indestruction of the myelin covering of nerve cells, particularly of thebrain and spinal cord. As used herein, “MS” includes all of theclassifications of MS known in the art, including, but not limitedRelapsing-remitting (RRMS) (typically characterized by partial or totalrecovery after attacks (also called exacerbations, relapses, orflares)), Secondary progressive (SPMS) (generally characterized by fewerrelapses, with an increase in disability and symptoms), and Primaryprogressive (PPMS) (generally characterized by progression of symptomsand disability without remission).

“Alzheimer's disease” or “AD” are terms understood in the art and usedherein to denote a progressive neurodegenerative disease characterizedby dementia and defined by the American Psychiatric Association (in DSMIV) as the development of multiple cognitive deficits that includesmemory impairment.

An “individual” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, farm animals, sportanimals, rodents, primates, and pets.

A “macrophage-associated neurodegenerative disease individual” or a“macrophage-associated neurodegenerative disease patient” is anindividual who is diagnosed as having a neurodegenerative disease or issuspected of having a neurodegenerative disease by demonstratingclinical symptoms of a neurodegenerative disease, which symptoms includepathologic macrophages in the patient's blood. A“non-macrophage-associated neurodegenerative disease individual” is anindividual who is not diagnosed as having, and not suspected of having,a macrophage-associated neurodegenerative disease.“Macrophage-associated neurodegenerative disorder” is specificallydefined herein to exclude cancer and autoimmune diseases.

An “ALS individual” or an “ALS patient” is an individual who isdiagnosed as having ALS or is suspected of having ALS by demonstratingALS-associated symptoms. A “non-ALS individual” is an individual who isnot diagnosed as having ALS or not suspected of having ALS. ALS andmethods of diagnosing ALS are known in the art and are discussed herein.

An “AD individual” or an “AD patient” is an individual who is diagnosedas having AD or is suspected of having AD by demonstrating AD-associatedsymptoms. A “non-AD individual” is an individual who is not diagnosed ashaving AD or not suspected of having AD. AD and methods of diagnosing ADare known in the art and are discussed herein.

An “MS individual” or an “MS patient” is an individual who is diagnosedas having MS or is suspected of having MS by demonstrating MS-associatedsymptoms. A “non-MS individual” is an individual who is not diagnosed ashaving MS or not suspected of having MS. MS and methods of diagnosing MSare known in the art and are discussed herein.

“Development” or “progression” of a disease, e.g., amacrophage-associated neurodegenerative disease such as ALS, of AD, orof MS, herein means initial manifestations and/or ensuing progression ofthe disorder. For example, development of ALS or of MS can be detectableand assessed using standard clinical techniques, such as nerve andmuscle biopsy and CNS scanning technologies such as MRI. However,development also refers to disease progression that may be undetectable.For purposes of this invention, development or progression refers to thebiological course of the disease state. “Development” includesoccurrence, recurrence, and onset. As used herein “onset” or“occurrence” of ALS, AD, or MS includes initial onset and/or recurrence.

As used herein, “delaying development” of a macrophage-associatedneurodegenerative disease a disease, such as ALS, AD or MS, means todefer, hinder, slow, retard, stabilize, and/or postpone development ofone or more symptoms, of the disease, including decreasing the rate atwhich the patient's disease progresses (e.g., to shift the patient fromrapidly progressing disease to a more slowly progressing disease). Thisdelay can be of varying lengths of time, depending on the history of thedisorder and/or the medical profile of the individual being treated. Asis evident to one skilled in the art, a sufficient or significant delaycan, in effect, encompass prevention, in that the individual does notdevelop detectable disease. A method that “delays” development ofdisease is a method that reduces the extent of the disease in a giventime frame, when compared to not using the method. Such comparisons aretypically based on clinical studies, using a statistically significantnumber of subjects, although this knowledge can be based upon anecdotalevidence. “Delaying development” can mean that the extent and/orundesirable clinical manifestations are lessened and/or time course ofthe progression is slowed or lengthened, as compared to notadministering the agent. Thus the term also includes, but is not limitedto, alleviation of symptoms, diminishment of extent of disease,stabilized (i.e., not worsening) state of disease, delay or slowing ofdisease progression, and remission (whether partial or total) whetherdetectable or undetectable.

As used herein, “biological sample” encompasses a variety of sampletypes obtained from an individual and can be used in a diagnostic ormonitoring assay. The definition encompasses blood and other liquidsamples of biological origin, solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom, and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such as proteinsor polynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, serum, plasma, biological fluid, and tissue samples. Generally,the sample will be, or be derived from, peripheral blood and as such isa “blood sample”. In some cases, the blood will have been enriched for amacrophage fraction, by using, for example, glass or plastic adherence.

A “blood sample” is a biological sample which is derived from blood,preferably peripheral (or circulating) blood. A blood sample may be, forexample, whole blood, plasma or serum.

As used herein, an “effective amount” (e.g., of an agent) is an amount(of the agent) that produces a desired and/or beneficial result. Aneffective amount can be administered in one or more administrations. Ingeneral, an effective amount is an amount sufficient to decrease thelevel of abnormal macrophages (pathologic macrophages) in amacrophage-associated neurodegenerative disease patient or derived froma macrophage-associated neurodegenerative disease individual. In someembodiments, an effective amount is an amount sufficient to decrease thelevel of abnormal macrophages in an ALS patient or derived from an ALSindividual. In other embodiments, an effective amount is an amountsufficient to decrease the level of abnormal macrophages in an MSpatient or derived from an MS individual. In other embodiments, aneffective amount is an amount sufficient to decrease the level ofabnormal macrophages in an AD patient or derived from an AD individual.An “amount sufficient to decrease the level of abnormal macrophages”preferably is able to decrease the level of abnormal macrophages by atleast about 25%, preferably at least about 50%, more preferably at leastabout 75%, and even more preferably at least about 90%. Such a decreasemay have desirable concomitant effects, such as to palliate, ameliorate,stabilize, reverse, slow or delay progression of disease, delay and/oreven prevent onset of disease.

In other embodiments, “amount sufficient to decrease the level of HLA-DRexpression by CD14+ cells” preferably is able to decrease the level ofHLA-DR expression by at least about 25%, preferably at least about 50%,more preferably at least about 75%, and even more preferably at leastabout 90%. Such a decrease may have desirable concomitant effects, suchas to palliate, ameliorate, stabilize, reverse, slow or delayprogression of disease, delay and/or even prevent onset of disease.

As used herein, decreasing the “level of abnormal macrophages” generallymeans decreasing the population number of abnormal macrophages oractivated monocytes and/or decreasing the level of HLA-DR expression ina population of CD14+ cells. In various embodiments, the level ofabnormal macrophages can be assayed by determining the percentage ofCD16+ cells in a population of CD14+ cells and/or the number ofCD14+/CD16+ cells in the biological sample. It is understood that anabsolute level need not be determined; an observation of a relativelevel of abnormal macrophages is sufficient.

“Modulating” macrophage proliferation means that the level or rate ofproliferation is altered when compared to not administering an agentthat changes macrophage proliferation. For example, “modulating”macrophage proliferation through use of chlorite-containing compositionmeans that the level of proliferating macrophages or the rate ofproliferation is altered when compared to not administering the agent.Preferably, “modulating” macrophage proliferation means a change in thelevel of proliferating macrophages or the rate of macrophageproliferation of at least 25%, preferably at least 50%, more preferablyat least 75%, and even more preferably at least 90%. Generally, forpurposes of this invention, “modulating” macrophage proliferation meansthat the level of proliferating macrophages or the rate of proliferationis decreased when compared to the same parameter in that individual whenno agent is administered. However, during the course of therapy, forexample, it may be desirable to increase the level of proliferatingmacrophages or the rate of proliferation from a previously measuredlevel. The degree of modulation may be assessed by measurement ofmacrophage proliferation, which will be discussed below, and generallyentails detecting a proliferation marker(s) in a macrophage populationor uptake of certain substances such as BrdU or 3H-thymidine (whichwould provide a quantitative measure of proliferation). Further, it ispossible that, if the macrophages are proliferating due to a geneticalteration (such as transposition, deletion, or insertion), thisalteration could be detected using standard techniques in the art, suchas RFLP (restriction fragment length polymorphism).

“Treatment” or “treating” as used herein means any therapeuticintervention in a subject, usually a mammalian subject, generally ahuman subject, including: (i) prevention, that is, causing overtclinical symptoms not to develop, e.g., preventing disease progressionto a harmful state; (ii) inhibition, that is, arresting the developmentor further development of clinical symptoms, e.g., mitigating existingclinical symptoms; and/or (iii) relief, that is, causing the regressionof clinical symptoms, e.g., causing relief from clinical symptoms.

Exemplary clinical symptoms of ALS include muscle weakness, musclewasting, muscle cramping, muscle twitching, slurred or slow speech,difficulty swallowing, and slow, uncoordinated movements. Furtherexemplary clinical symptoms of ALS include those detectable in abiological sample obtained from a subject having or suspected of havingALS, e.g., increased CD4:CD8 cell ratio compared to normal, decreasednumber of CD14+ cells compared to normal, increased expression of HLA-DRon CD14+ cells compared to normal CD14+ cells, increased levels ofactivated monocytes or macrophages compared to normal, the presence ofproliferating macrophages, and decreased serum IgG and/or IgM comparedto normal, where “normal” as used herein means a subject unaffected byALS or cells from such an unaffected subject. “Treating” thusencompasses achieving a decrease in one or more clinical symptoms, whichdecrease may have desirable concomitant effects, such as to palliate,ameliorate, stabilize, reverse, slow or delay progression of disease,delay and/or even prevent onset of disease.

Exemplary clinical symptoms of AD include mild forgetfulness, includingtrouble remembering recent events, activities, or the names of familiarpeople or things; difficulty in solving simple math problems; troubleremembering how to do simple tasks (e.g., brushing teeth or combinghair); inability to think clearly; difficulty speaking, understanding,reading, or writing; and anxiety or aggressiveness, or tendency towander away from home.

Exemplary clinical symptoms of MS include fatigue (also referred to asMS lassitude), muscle fatigue, paresthesias, difficulty in walkingand/or balance problems, abnormal sensations such as numbness,prickling, or “pins and needles”, pain, bladder dysfunction, boweldysfunction, changes in cognitive function (including problems withmemory, attention, concentration, judgment, and problem-solving),dizziness and vertigo, emotional problems (e.g., depression), sexualdysfunction, and vision problems. Severe cases can involve partial orcomplete paralysis. (such as blurred or double vision, red-green colordistortion, or even blindness in one eye). Other symptoms includeheadache, hearing loss, itching, seizures, spasticity, speech andswallowing disorders, and tremors. Further exemplary clinical symptomsof MS include those detectable in a biological sample obtained from asubject having or suspected of having MS, e.g., increased CD4:CD8 cellratio compared to normal, decreased number of CD14+ cells compared tonormal, increased expression of HLA-DR on CD14+ cells compared to normalCD14+ cells, increased levels of activated monocytes or macrophagescompared to normal, the presence of proliferating macrophages, anddecreased serum IgG and/or IgM compared to normal, where “normal” asused herein means a subject unaffected by MS or cells from such anunaffected subject. “Treating” thus encompasses achieving a decrease inone or more clinical symptoms, which decrease may have desirableconcomitant effects, such as to palliate, ameliorate, stabilize,reverse, slow or delay progression of disease, delay and/or even preventonset of disease.

The terms “subject” and “patient” mean a member or members of anymammalian or non-mammalian species that may have a need for thepharmaceutical methods, compositions and treatments described herein.Subjects and patients thus include, without limitation, primate(including humans), canine, feline, ungulate (e.g., equine, bovine,swine (e.g., pig)), avian, and other subjects. Humans and non-humananimals having commercial importance (e.g., livestock and domesticatedanimals) are of particular interest.

“Mammal” means a member or members of any mammalian species, andincludes, by way of example, canines; felines; equines; bovines; ovines;rodentia, etc. and primates, particularly humans. Non-human animalmodels, particularly mammals, e.g. primate, murine, lagomorpha, etc. maybe used for experimental investigations.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of compounds ofthe present invention calculated in an amount sufficient to produce thedesired effect in association with a pharmaceutically acceptableexcipient (e.g., pharmaceutically acceptable diluent, carrier orvehicle).

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes an excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. “A pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

Chlorite and Administration Thereof

The source of chlorite ions for administration of chlorite according tothe invention can be provided in a variety of forms. For example,chlorite can be administered as a chlorite salt (e.g., alkali metalsalt, e.g., sodium chlorite, potassium chlorite, and the like) or amixture of chlorite salts, where the chlorite salts are preferablypharmaceutically acceptable. In addition or alternatively, chlorite canbe administered as a matrix of chlorite ions, e.g., described in U.S.Pat. No. 4,507,285. In one embodiment, the chlorite ions are provided ina compositions having the general formula

ClO₂ ×nO₂

wherein “n” can be a value of about 0.1-0.25. Such agents can have an O₂band at 1562 cm⁻¹ in the Raman spectrum and an O-O interval of 123 pm.Production of such agents is known in the art, see, e.g., U.S. Pat. No.4,507,285.

In one embodiment, the method of treatment involves administration of anaqueous solution of a product known as “tetrachlorodecaoxygen anioncomplex”, commonly abbreviated as “TCDO”. Production of TCDO is wellknown, see, e.g., Example 1 of U.S. Pat. No. 4,507,285.

As appropriate, agents that provide a source of chlorite ions can beadministered in a free base or free acid form (that is, as the freecompound and not as a salt). Additionally, any pharmaceuticallyacceptable salt(s) of the compound(s) can also be used. Pharmaceuticallyacceptable salts are those salts which retain the biological activity ofthe free compounds and which are not biologically or otherwiseundesirable. As appropriate, stereoisomers of the compounds disclosedcan also be used in the invention, including diastereomers andenantiomers, as well as mixtures of stereoisomers, including, but notlimited to, racemic mixtures. Unless stereochemistry is explicitlyindicated in a structure, the structure is intended to embrace allpossible stereoisomers of the compound depicted.

Formulations

Chlorite can be provided in any suitable formulation, which can beselected according to the desired route of administration.

U.S. Pat. No. 4,725,437 describes an aqueous solution of a chemicallystabilized chlorite matrix suitable for intravenous administration in adosed amount of about 6.2×10⁻⁶ mole of ClO₂ ⁻⁻ to 9.3×10⁻⁵ mole of ClO₂⁻⁻ per kg of body weight in humans and non-human animals. The solutioncontains the chlorite matrix in a concentration of about 12 to 72micromol of ClO₂ ⁻⁻ per ml. Further chlorite formulations are describedin U.S. Pat. Nos. 4,507,285 and 4,725,437.

Formulations of TCDO are of particular interest in the presentinvention. WF10 is a TCDO formulation of particular interest in thepractice of the invention. WF10, also known as Oxoferin (Oxo ChemieGmbH, Fort Worth, Tex.), is available commercially. Other formulationsof TCDO are within the scope of this invention.

Chlorite-containing compositions, such as TCDO, can be formulated forparenteral or enteral administration, generally parenteraladministration. Accordingly, formulations of chlorite are suitable forparenteral, topical, or transdermal administration, usually intravenous,intramuscular, or subcutaneous administration, and may be suitable foradministration by bolus injection, sustained release (includingcontrolled release), infusion, and the like. Administration by infusion(e.g., by subcutaneous or intravenous infusion) is of interest, as isadministration in the form of suppositories. Additional agents andtherapies

Chlorite can be administered alone or in various combinations. Whereadministered in combination, chlorite can be administered in conjunctionwith other agents, particularly those suitable for protective,palliative or supportive care of the subject. The phrase “in conjunctionwith” means that an agent is administered prior to, concurrently, orafter other substance or therapy. Examples of agents for administrationin conjunction with an agent include, but are not limited to, riluzole.Other agents for administration in conjunction with chlorite includeagents for control of symptoms of a macrophage-associatedneurodegenerative disorder, such as ALS, AD or MS symptoms. Furtherexemplary agents for administration in conjunction with chloriteaccording to the invention include, but are not limited to, baclofen,diazepam, trihexyphenidyl and/or amitriptyline. Chlorite can also beadministered in conjunction with non-drug therapy (e.g., physical and/oroccupational therapy, massage, and the like).

In one embodiment, the composition does not contain an amount of anotheranti-proliferative agent, such as a polyamine analog, effective todecrease the level of abnormal macrophages in a macrophage-associatedneurodegenerative disorder patient, such as an ALS, AD, or MS patient(e.g., as compared to prior to therapy). For example, TCDO has beendescribed for administration in combination therapy withanti-proliferative agents where TCDO is administered in an amounteffective to promote macrophage phagocytosis to facilitate delivery ofthe anti-proliferative agent to the macrophage. The present inventioncontemplates that chlorite ions (e.g., as a pharmaceutically acceptablesalt or in a stabilized matrix, such as in TCDO) are administered to amacrophage-associated neurodegenerative disorder patient, such as anALS, AD or MS patient so that the chlorite is the active ingredientpresent in the subject in an amount effective to facilitate treatment ofthe patient e.g., through reduction in proliferating/inappropriatelyactivated macrophages, and without the need for administration of forexample, a polyamine analog or other anti-proliferative agent inconjunction with chlorite.

Administration and Dosing

Chlorite formulations are generally dosed in vivo corresponding to thebody weight of the subject. Due to the continuous breakdown of theactive agent in the blood, the agent is normally administered at regularintervals. Those of skill in the art will readily appreciate that actualdosages and regimen will vary as a function of the agent, formulation,the severity of the symptoms, the susceptibility of the subject totreatment and/or side effects, and the like. Dosages are readily androutinely determinable by those of skill in the art by a variety ofmeans.

Exemplary doses of chlorite-containing formulations can vary betweenabout 0.1 ml/kg to about 1.5 ml/kg, preferably, about 0.5 ml/kg of bodyweight and at a concentration of about 40 to about 80 mMol ClO₂ ⁻ perliter, usually about 60 mMol ClO₂ ⁻ per liter, respectively. In the caseof TCDO, a dose finding phase I/II study evaluating WF-10 administeredintravenously and involving 48 patients established a maximum dose ofapproximately 0.5 ml/kg. Other suitable doses may be approximately 0.25ml/kg.

The regimen of administration (e.g., dose combined with frequency ofadministration) will generally involve administration in an amount andat a frequency to provide for a desired effect, e.g., administration ofan amount effective to provide for improvement in one or more symptomsof a macrophage-associated neurodegenerative disorder patient, such asone or more ALS, AD or MS symptoms. For example, chlorite can beadministered for 2, 3, 4, 5, 6, 7, 8, 9, 10 or more consecutive days,which administration period may be reinitiated after 1, 2, 3 or moreweeks following the last dose. In one exemplary embodiment, a WF-10regimen comprises 5 consecutive days of treatment every 3 weeks.

In one embodiment, chlorite is administered so as to effect modulationof macrophage proliferation, e.g., alteration of the level ofproliferating macrophages or the rate of macrophage proliferationcompared to in the absence of agent administration, and/or to effectmodulation of inappropriate macrophage activation. An effective amountof chlorite is determined by, for example, comparing the level (ornumber) of promacs, before and during treatment, with a downward trendin the number of promacs generally being consistent with a positiveeffect. In one embodiment, chlorite is administered so as to effect achange in the level of proliferating macrophages or the rate ofmacrophage proliferation of at least 25%, preferably at least 50%, morepreferably at least 75%, and even more preferably at least 90%. Thedegree of modulation may be assessed by measurement of macrophageproliferation as described in the art, and generally entails detecting aproliferation marker(s) in a macrophage population or uptake of certainsubstances such as BrdU or 3H-thymidine (which would provide aquantitative measure of proliferation) (see, e.g., U.S. Publication No.20030175832). Such a decrease may have desirable concomitant effects,such as to palliate, ameliorate, stabilize, reverse, slow and/or delayprogression of disease, delay or even prevent onset of disease.

Methods for detecting proliferating or inappropriately activatedmacrophages and determining macrophage proliferation rates are known inthe art. For example, proliferating macrophages may be detected byassaying cell proliferative markers, such as PCNA, Ki67 or uptake ofbromodeoxyuridine (BrdU) or 3H-thymidine. These markers are distinctfrom those that identify only “activated” macrophages (as opposed toproliferating macrophages), such as CD69 and CD25. The cellular subsetrepresenting macrophages may in turn be identified by detection ofcertain cell specific markers, such as CD14, CD68, CD16, or nonspecificesterase. Detection of these cell-type and/or proliferative markers usemethods standard in the art, such as staining techniques and FACSsorting and analysis.

In another embodiment, chlorite is administered to as to effect adecrease in the level (e.g., number) of pathologic macrophages, e.g., toeffect a decrease in the level of CD14+ monocytes, preferably activatedCD14+ monocytes, in a patient with a macrophage-associatedneurodegenerative disorder (e.g., a patient with ALS, with AD, or withMS). In this embodiment, chlorite is administered in an amountsufficient to decrease the level of (e.g., number of) CD14+ monocytes,preferably activated CD14+ monocytes and/or CD14+ monocytes withelevated HLA-DR expression and/or the number of CD14+/CD16+ cells and/orthe percentage of CD16+ cells in a population of CD14+ cells in theindividual (i.e., an effective amount). An effective amount of chloriteis determined by, for example, comparing the level of number of CD14+monocytes, preferably activated CD14+ monocytes, before and duringtreatment, with a downward trend of number of CD14+ monocytes generallybeing consistent with a positive effect. An “amount sufficient todecrease the number of CD14+ monocytes” preferably is able to decreasethe number of CD14+ monocytes by at least about 25%, preferably at leastabout 50%, more preferably at least about 75%, and even more preferablyat least about 90%. Methods for assessing levels of CD14+ monocytes,activated CD14+ monocytes, CD14+ monocytes with elevated HLA-DRexpression, CD14+/CD16+ cells and the percentage of CD16+ cells in apopulation of CCD14+ are known in the art (see, e.g., U.S. PublicationNo. 20030175832). Such a decrease may have desirable concomitanteffects, such as to palliate, ameliorate, stabilize, reverse, slowand/or delay progression of disease, delay or even prevent onset ofdisease.

Levels of pathologic macrophages (proliferating/inappropriate activatedmacrophages (promacs)), macrophage proliferation rate, CD14+ cells,HLA-DR expression, and the like as set out above can be compared to alevel from the same individual measured at a different time and/or underdifferent conditions (such as before treatment, different dose, etc.),and/or to a mean or median level determined for a non-diseased standard(e.g., non-macrophage-associated neurodegenerative disorder patient,such as a non-ALS, non-AD, or non-MS, as appropriate), for example froman unaffected individual (e.g., non-macrophage-associatedneurodegenerative disorder individual or individuals; a non-ALSindividual or non-ALS individuals; or non-AD individual or non-ADindividuals; or non-MS individual or non-MS individuals).

For example, an HLA-DR expression level may be compared to an HLA-DRlevel from the same individual measured at a different time and/or underdifferent conditions (such as before treatment, different dose, etc.).In some embodiments, an HLA-DR expression level is compared to a mean ormedian level of HLA-DR expression determined on a population of CD14+cells from a non-diseased (e.g., non-ALS, non-AD, or non-MS) standard,for example from a non-ALS individual or non-ALS individuals, or non-MSindividual or non-MS individuals, or non-AD individual or non-ADindividuals). A finding of HLA-DR expression level of greater than about1.4 fold that of the non-diseased standard is indicative of an elevatedlevel of HLA-DR expression in the individual. Generally, a finding ofHLA-DR expression level of greater than about 1.5 fold, greater thanabout 1.6 fold, greater than about 1.7 fold, greater than about 1.8fold, greater than about 1.9 fold, greater than about 2.0 fold, greaterthan about 5.0 fold, or greater than about 10 fold that of anon-diseased standard is indicative of an elevated level of HLA-DRexpression in the individual. Thus, decreasing HLA-DR expression in amacrophage-associated neurodegenerative disorder subject (e.g., an ALSsubject, an AD subject, or an MS subject) so as to more closelyapproximate an HLA-DR expression level in a non-diseased subject (e.g.,non-ALS, non-AD, or non-MS subject) is of interest in the presentinvention.

In another example, the number of CD14+/CD16+ cells or the percentage ofCD16+ cells in a population of CD14+ cells in a sample from amacrophage-associated neurodegenerative disorder subject (e.g., an ALSsubject, an AD subject, or an MS subject) is compared to a mean ormedian level of CD14+/CD16+ cells in a biological sample from anon-disease (e.g., non-ALS, non-AD, or non-MS) standard, for examplefrom a non-ALS individual or non-ALS individuals; or non-AD individualor non-AD individuals or non-MS individual or non-MS individuals. Afinding of a percentage of CD16+ cells in a population of CD14+ cellsand/or the number of CD14+/CD16+ cells in a sample of greater than about1.5 fold, greater than about 1.6 fold, greater than about 1.7 fold,greater than about 1.8 fold, greater than about 1.9 fold, greater thanabout 2.0 fold, greater than about 3.0 fold, greater than about 4.0fold, greater than about 5.0 fold, or greater than about 10 fold that ofa non-diseased (non-ALS, non-AD, or non-MS) standard is indicative of anincreased number of CD14+/CD16+ cells in the individual. Thus, in oneembodiment, therapy according to the invention is provided so as todecrease the number of CD14+/CD16+ cells or the percentage of CD16+cells in a population of CD14+ cells so as to more closely approximatesuch in an appropriate non-diseased subject.

In general, therapy is monitored by following blood macrophageactivation, usually by following CD14/DR levels and the percentage ofCD14/16 positive cells as described above.

Kits with unit doses of the subject compounds, usually in injectabledoses, are provided. In such kits, in addition to the containerscontaining the unit doses will be an informational package insertdescribing the use and attendant benefits of chlorite in treating amacrophage-associated neurodegenerative disorder subject, such as ALS,AD, or MS. Preferred compounds and unit doses are those described hereinabove.

Subjects and Monitoring Therapy

In general, individuals suitable for therapy involving administration ofchlorite according to the invention include individuals who have beendiagnosed as having a macrophage-associated neurodegenerative disorder,are “afflicted with” a macrophage-associated neurodegenerative disorder(e.g., diagnosed as having, suffering from and/or displaying one or moreclinical symptoms), or who have been adjudged to be at high risk fordeveloping such a disorder. An “at risk” or “high risk” individual is anindividual who has a discrete and significant risk of developing amacrophage-associated neurodegenerative disorder. An “at risk” or “highrisk” individual may or may not have detectable disease, and may or maynot have displayed detectable disease prior to receiving the method(s)described herein. “High risk” (or “at risk”) denotes that an individualhas one or more so-called risk factors, which are measurable parametersthat correlate with development of disease. An individual having one ormore of these risk factors has a higher probability of developingdisease than an individual without these risk factor(s). These riskfactors include, but are not limited to, genetic (i.e., hereditary)considerations (including family history and genetic markers). It isunderstood that having only one risk factor can often indicate highrisk. The clinician, as one skilled in the art, has discretion todetermine whether treatment using an agent may be indicated for anindividual at risk. Exemplary a macrophage-associated neurodegenerativedisorders includes ALS, AD, and MS.

In one embodiment, individuals suitable for therapy involvingadministration of chlorite according to the invention includeindividuals who have been diagnosed as having ALS, are “afflicted with”ALS (e.g., diagnosed as having, suffering from and/or displaying one ormore clinical symptoms of) ALS, or who have been adjudged to be at highrisk for developing such a disorder. An “at risk” or “high risk”individual is an individual who has a discrete and significant risk ofdeveloping ALS. An “at risk” or “high risk” individual may or may nothave detectable disease, and may or may not have displayed detectabledisease prior to receiving the method(s) described herein. “High risk”(or “at risk”) denotes that an individual has one or more so-called riskfactors, which are measurable parameters that correlate with developmentof disease. An individual having one or more of these risk factors has ahigher probability of developing disease than an individual withoutthese risk factor(s). These risk factors include, but are not limitedto, genetic (i.e., hereditary) considerations (including family historyand genetic markers). It is understood that having only one risk factorcan often indicate high risk. The clinician, as one skilled in the art,has discretion to determine whether treatment using an agent may beindicated for an individual at risk.

Exemplary clinical symptoms of ALS include muscle weakness, musclewasting, muscle cramping, muscle twitching, slurred or slow speech,difficulty swallowing, and slow, uncoordinated movements. Furtherexemplary clinical symptoms of ALS include those detectable in abiological sample obtained from a subject having or suspected of havingALS, e.g., increased CD4:CD8 cell ratio compared to normal, decreasednumber of CD14+ cells compared to normal, increased expression of HLA-DRon CD14+ cells compared to normal CD14+ cells, increased levels ofactivated monocytes or macrophages compared to normal, the presence ofproliferating macrophages, and decreased serum IgG and/or IgM comparedto normal, where “normal” as used herein means a subject unaffected byALS or cells from such an unaffected subject.

In another embodiment, individuals suitable for therapy involvingadministration of chlorite according to the invention includeindividuals who have been diagnosed as having MS, are “afflicted with”MS (e.g., diagnosed as having, suffering from and/or displaying one ormore clinical symptoms of) MS, or who have been adjudged to be at highrisk for developing such a disorder. An “at risk” or “high risk”individual is an individual who has a discrete and significant risk ofdeveloping MS. An “at risk” or “high risk” individual may or may nothave detectable disease, and may or may not have displayed detectabledisease prior to receiving the method(s) described herein. “High risk”(or “at risk”) denotes that an individual has one or more so-called riskfactors, which are measurable parameters that correlate with developmentof disease. An individual having one or more of these risk factors has ahigher probability of developing disease than an individual withoutthese risk factor(s). These risk factors include, but are not limitedto, genetic (i.e., hereditary) considerations (including family historyand genetic markers). It is understood that having only one risk factorcan often indicate high risk. The clinician, as one skilled in the art,has discretion to determine whether treatment using an agent may beindicated for an individual at risk.

Exemplary clinical symptoms of MS include those detectable in abiological sample obtained from a subject having or suspected of havingMS, e.g., increased CD4:CD8 cell ratio compared to normal, decreasednumber of CD14+ cells compared to normal, increased expression of HLA-DRon CD14+ cells compared to normal CD14+ cells, increased levels ofactivated monocytes or macrophages compared to normal, the presence ofproliferating macrophages, and decreased serum IgG and/or IgM comparedto normal, where “normal” as used herein means a subject unaffected byMS or cells from such an unaffected subject.

In another embodiment, individuals suitable for therapy involvingadministration of chlorite according to the invention includeindividuals who have been diagnosed as having AD, are “afflicted with”AD (e.g., diagnosed as having, suffering from and/or displaying one ormore clinical symptoms of) AD, or who have been adjudged to be at highrisk for developing such a disorder. An “at risk” or “high risk”individual is an individual who has a discrete and significant risk ofdeveloping AD. An “at risk” or “high risk” individual may or may nothave detectable disease, and may or may not have displayed detectabledisease prior to receiving the method(s) described herein. “High risk”(or “at risk”) denotes that an individual has one or more so-called riskfactors, which are measurable parameters that correlate with developmentof disease. An individual having one or more of these risk factors has ahigher probability of developing disease than an individual withoutthese risk factor(s). These risk factors include, but are not limitedto, genetic (i.e., hereditary) considerations (including family historyand genetic markers). It is understood that having only one risk factorcan often indicate high risk. The clinician, as one skilled in the art,has discretion to determine whether treatment using an agent may beindicated for an individual at risk.

Exemplary clinical symptoms of AD include mild forgetfulness, includingtrouble remembering recent events, activities, or the names of familiarpeople or things; difficulty in solving simple math problems; troubleremembering how to do simple tasks (e.g., brushing teeth or combinghair); inability to think clearly; difficulty speaking, understanding,reading, or writing; and anxiety or aggressiveness, or tendency towander away from home. Further exemplary clinical symptoms of AD includethose detectable in a biological sample obtained from a subject havingor suspected of having AD, e.g., increased CD4:CD8 cell ratio comparedto normal, decreased number of CD14+ cells compared to normal, increasedexpression of HLA-DR on CD14+ cells compared to normal CD14+ cells,increased levels of activated monocytes or macrophages compared tonormal, the presence of proliferating macrophages, and decreased serumIgG and/or IgM compared to normal, where “normal” as used herein means asubject unaffected by AD or cells from such an unaffected subject.

Monitoring Therapy

Chlorite-based therapy according to the invention can be monitored, anddosages and regimen adjusted accordingly, by assessing the effect oftherapy upon one or more clinical symptoms. In general, an effectiveamount of chlorite is a dose or doses that provide for an improvement inone or more clinical symptoms in the subject.

For example, since elevated HLA-DR expression on CD14+ cells and/orincreased numbers of CD14+/CD16+ cells and/or the percentage of CD16+cells in a population of CD14+ cells is associated with amacrophage-associated neurodegenerative disorder (e.g., ALS, AD, MS),monitoring these levels can be used to facilitates assessment of initialresponsiveness to therapy and/or efficacy, as well as the appropriatedosage of the therapy. Similarly, since elevated HLA-DR expression onCD14+ cells and/or increased numbers of CD14+/CD16+ cells and/or thepercentage of CD16+ cells in a population of CD14+ cells is associatedwith MS, monitoring these levels can be used to facilitates assessmentof initial responsiveness to therapy and/or efficacy, as well as theappropriate dosage of the therapy.

It is understood that monitoring therapy means that symptoms areassessed at different times and are compared over time. Where assessmentof a clinical symptom requires analysis of a biological sample, suchbiological sample(s) are generally obtained at different times, forexample, during application of therapy, and are compared, either witheach other, a control, and/or a desired value. Methods for monitoringALS therapy through assessment of biological samples is described in,for example, U.S. Publication No. 20030175832.

For example, therapy for a macrophage-associated neurodegenerativedisorder, such as ALS, AD or MS therapy can be monitored by determiningthe level of HLA-DR expression by CD14+ cells from peripheral blood. Inanother embodiment, monitoring therapy includes the step of determiningthe level of CD14+ cells expressing elevated HLA-DR in a blood sample,preferably peripheral blood. In another embodiment, monitoring therapyincludes the step of determining the percentage of CD16+ cells in thepopulation of CD14+ cells in a blood sample, preferably peripheralblood. In another embodiment, monitoring therapy includes the step ofdetermining the number of CD14+/CD16+ cells in a blood sample,preferably peripheral blood. In another embodiment, the level ofabnormal macrophages (in various embodiments, the level of CD14+ cellsexpressing elevated HLA-DR; the percentage of CD16+ cells in thepopulation of CD14+ cells and/or the number of CD14+/CD16+ cells) in ablood sample determined during and/or at completion of the therapy isgenerally compared with the level in a control sample and/or with adesired value. In another embodiment, monitoring therapy also includesthe step of measuring proliferation of the abnormal macrophages.

In another embodiment, therapy for a macrophage-associatedneurodegenerative disorder, such as ALS, AD or MS therapy is monitoredby assessing the level of abnormal macrophages in a sample taken at aparticular time from a patient undergoing the therapy and/or a sampletaken after or at completion of the therapy is generally compared withthe level in a sample taken from the patient prior to the therapy and/orwith the level in a sample taken from the patient at a different timepoint in the therapy. For example, a decrease in the level of abnormalmacrophages in the sample taken during therapy as compared to the sampletaken prior to or at an earlier time point in therapy would generally beconsistent with a positive effect of the therapy.

In another embodiment, therapy according to the invention is monitoredby assessing the level of abnormal macrophages is assessed by thedetermining the level of HLA-DR expression by CD14+ cells from a bloodsample, such as a peripheral blood sample. For example, the effect of atherapy is determined by comparing the level of HLA-DR expression byCD14+ cells in peripheral blood before and during treatment, with adownward trend in HLA-DR expression generally being consistent with apositive effect.

In another embodiment, therapy according to the invention is monitoredby assessing the level of pathologic macrophages, e.g., by assessing thelevel of abnormal macrophages is assessed by the determining thepercentage of CD16+ cells in the population of CD14+ cells from a bloodsample, such as a peripheral blood sample. For example, the effect of atherapy is determined by comparing the percentage of CD16+ cells in thepopulation of CD14+ cells in peripheral blood before and duringtreatment, with a downward trend in the percentage of CD14+/CD16+ cellsgenerally being consistent with a positive effect.

In another embodiment, therapy according to the invention is monitoredby assessing the level of pathologic macrophages, e.g., by assessing thelevel of abnormal macrophages is assessed by the determining the numberof CD14+/CD16+ cells in a blood sample, such as a peripheral bloodsample. For example, the effect of a therapy is determined by comparingthe number of CD14+/CD16+ cells in peripheral blood before and duringtreatment, with a downward trend in the number of CD14+/CD16+ cellsgenerally being consistent with a positive effect.

Kits

The invention also contemplates kits with unit doses of a source ofchlorite ions, e.g., a chlorite salt (e.g., alkali metal salt, e.g.,sodium chlorite, potassium chlorite, and the like); a mixture ofchlorite salts; a matrix of chlorite ions, e.g., a compositions havingthe general formula ClO₂×nO₂, wherein “n” can be a value of about0.1-0.25; e.g., TCDO. In general such unit doses are in injectabledosage forms, more particularly dosage forms suitable for infusion. Insuch kits, in addition to the containers containing the unit doses willbe an informational package insert describing the use and attendantbenefits of chlorite in treating a macrophage-associatedneurodegenerative disorder subject, such as ALS, AD, or MS. Optionally,the kit includes information relating to identification of patientshaving a macrophage-associated neurodegenerative disease and monitoringof therapy of such patients (e.g., information relating to assessment ofpathologic macrophages, e.g., proliferating macrophages, activatedmacrophages).

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Methods and Materials

The following methods and materials were used in the Examples set outbelow.

Subjects

Forty patients with ALS (mean age±SD, 59.5±13.3 yr), diagnosed by ElEscorial criteria (Brooks et al. El Escorial World Federation ofNeurology criteria for the diagnosis of amyotrophic lateral sclerosis.Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis ofthe World Federation of Neurology Research Group on NeuromuscularDiseases and the El Escorial “Clinical limits of amyotrophic lateralsclerosis” workshop contributors. J. Neurol. Sci. 124(suppl):96-107) atthe Forbes Norris MDA/ALS Research Center (San Francisco, Calif., USA)had blood drawn in accordance with the CPMC and UCSF committees on humanresearch guidelines, coordinated by the UCSF AIDS and Cancer SpecimenResource (ACSR) program. Revised ALS Functional Rating Scale (ALSFRS-R),scored 0-48, used to evaluate overall functional status in clinicaltrials as well as in clinical practice (Cedarbaum et al. 1999. TheALSFRS-R: a revised ALS functional rating scale that incorporatesassessments of respiratory function. J. Neurol. Sci. 169:13-21), wereused to evaluate each patient's clinical status and were updated withina month of blood testing.

The forty patients consisted of 26 men (age range, 34-87 yr; meanage±SD, 58.0±14.0 yr) and 14 women (age range, 40-77 yr; mean age±SD,62.4±11.7 yr). They had had ALS for 4 to 93 months with a range ofALSFRS-R scores of 8 to 43. Only two patients had familial ALS (fALS)and 38 patients were diagnosed with sporadic ALS (sALS). Demographicinformation on ALS patients (abbreviated in the table as “Pt”) whosespecimens were studied is shown in Table 1, in which thirteen ofpatients were using various anti-inflammatory medications with standarddose (Celebrex, Vioxx, Naproxyn, Excedrin), and 31 patients were takingriluzole (50 mg twice daily); ten patients received both medications.

TABLE 1 Clinical summary Pt Duration Pt Age Pt Disease Therapy ofIllness ALSFRS-R ID# (yrs) Sex Form riluzole^(A) NSAIDS^(B) (Months)Score Pt 1 76 F sALS No No 46 19 Pt 2 59 M sALS Yes No 15 30 Pt 3 77 FsALS Yes No 37 34 Pt 4 73 F fALS Yes No 31 20 Pt 5 57 M sALS YesCelebrex 19 34 Pt 6 63 F sLAS Yes Vioxx 10 N/A Pt 7 75 F sALS No No 7833 Pt 8 64 M sALS Yes No 43 13 Pt 9 58 F sLAS Yes No 42 28 Pt 10 72 MsALS Yes No 18 26 Pt 11 40 F sALS Yes No 12 28 Pt 12 58 M sLAS YesCelebrex 21 18 Pt 13 55 M sALS Yes No 85 N/A Pt 14 82 M sALS No Celebrex4 N/A Pt 15 67 M sLAS Yes Celebrex 45 20 Pt 16 79 M sALS Yes No 14 15 Pt17 49 M sALS No No 88 16 Pt 18 60 M sLAS No No 18 32 Pt 19 49 M sALS YesN/A 26 29 Pt 20 37 M sALS Yes Celebrex 82  8 Pt 21 70 M sALS Yes No 29N/A Pt 22 49 M sALS Yes No 14 39 Pt 23 41 F sALS Yes No 33 37 Pt 24 58 MsALS Yes No 20 32 Pt 25 30 M sLAS No No 24 35 Pt 26 65 M sLAS Yes No 1842 Pt 27 41 M sALS Yes No 43 43 Pt 28 58 M sALS Yes Celebrex 25 26 Pt 2966 F sLAS Yes No 6 39 Pt 30 63 F sALS Yes Excedrin 18 34 Pt 31 65 M sALSYes Celebrex 33 34 Pt 32 52 F fALS No Celebrex 8 26 Pt 33 34 M sALS YesCelebrex 41 15 Pt 34 47 M sLAS Yes No 17 38 Pt 35 62 M sALS Yes No 57 43Pt 36 87 M sALS No No 93 25 Pt 37 64 F sLAS Yes Celebrex 45 35 Pt 38 60F sALS Yes No 27 23 Pt 39 65 F sLAS Yes No 27 30 Pt 40 53 M sALS NoNaproxyn 45 37 ^(A)50 mg twice daily. ^(B)Standard dose.

37 normal control blood samples (mean age±SD, 41.8±9.2 yr) were obtainedfrom blood draws at Stanford University Blood Center and processed in asimilar manner to the ALS patient blood specimens. They consisted of 21men (age range, 25-61 yr; mean age±SD, 43.5±8.6 yr) and 16 women (agerange, 25-59 yr; mean age±SD, 35.9±9.7 yr). Control samples for IgG andIgM studies consisted of plasma from 80 blood donors and were alsoobtained from the Stanford University Blood Center.

Flow Cytometry

10 ml of peripheral blood was drawn from each patient and normalcontrols into heparinized tubes and transferred to the laboratory atroom temperature for same day immunologic studies. Cellular immunologicactivation was evaluated by quantitating levels of CD38 on T-cellsubsets and HLA-DR on CD14 cells. CD16 (Fc gamma III receptor)expression on CD14 cells was used as another marker for monocytedifferentiation and has been an antigen associated with cytokineexpression patterns characteristic of tissue macrophages(Ziegler-Heitbrock et al. 1993. Eur. J. Immunol. 23:2053-2058;Frankenberger et al. 1996. Blood. 87:373-377). The monocyte granularityassociated with its differentiation was measured by CD14-associated“backgating” on side light-scatter characteristics (SSC). Whole bloodwas stained with CD14-fluorescein isothiocyanate (FITC),CD16-phycoerythrin (PE) (DAKO, Carpinteria, Calif., USA), CD8-FITC,CD38-PE, HLA-DR-PE, and CD4-peridinin chlorophyll protein (PerCP)(Becton-Dickinson, San Jose, Calif., USA) for 15 minutes at roomtemperature. Negative controls consisted of aliquots stained withisotype IgG-FITC, IgG-PE, and IgG-PerCP; all staining was performed asper manufacturers specifications. Samples were then lysed with FACSLysing Solution (Becton-Dickinson) for 10 minutes at room temperaturefollowed by 0.1% sodium azide+PBS Ca++Mg++ free wash. The stained cellswere then resuspended in 1 ml of fixing solution (1% paraformaldehyde inPBS, with 0.1% sodium azide). Analysis was accomplished by acquisitionof data on a FACScan flow cytometer (Becton-Dickinson) with Cellquestsoftware where at least 20,000 cells were counted per analysis.

Detection of Serum IgG and IgM

Plasma from ALS patient blood was obtained by Percoll gradientcentrifugation, and was frozen at −70° C. until use. Standard ELISA fordetermination of serum antibody:

Anti-Human IgG Fab or anti-Human IgM (Sigma, St. Louis, Mo., USA) werecoated (100 mcl/well) into 96-well ELISA plates (Nunc, Roskilde,Denmark) by incubation for at least one hour at 37° C. The plates werewashed one time with TBS (150 mM NaCl, mM Tris-HCl, pH7.4), then blockedfor 30 minutes by addition of 150 mcl (microliters)/well of BLOTTO (TBSplus 0.1% Tween-20, 2.5% normal goat serum, 2.5% non fat dry milk) atroom temperature, with gentle rocking. ELISA plates were subsequentlywashed once (1×) with TBS. Serial dilutions of serum were added tocoated plates (duplicate wells each dilution, 100 mcl/well) and allowedto react for 90 minutes, room temperature. A standard calibration series(0 to 5 mcg/ml) for IgG and IgM (Sigma) was prepared, added to ELISAwells, and incubated in parallel. BLOTTO was used in all dilutions.Following the 90-minute incubation, all fluids were removed byaspiration, then all plates were washed 3× with TBS. Bound IgGantibodies were detected by adding 100 mcl/well of anti-Human IgGalkaline phosphatase-conjugate (Promega Corp., Madison, Wis., USA)diluted 1:10000 in BLOTTO. Bound IgM antibodies were detected by adding100 mcl/well of anti-Human IgM alkaline phosphatase-conjugate(Kirkegaard & Perry, Gaithersburg, Md., USA) diluted 1:5000 in BLOTTO.Antibody conjugates were incubated for 8one hour at room temperaturewith gentle agitation. Conjugates were removed by aspiration and plateswashed 4× with TBS. Development of color reaction was effected byaddition of 100 mcl of PNPP substrate (Sigma) to each well, followed byincubation for 20 minutes at room temperature. The optical density(O.D.) in each well was read at 405 nm. Any sera with exceptionally lowor high values were re-tested. Raw IgG and IgM values from ALS sampleswere multiplied by a conversion factor to account for the differentmeans of preparation from normal plasma.

Statistical Analysis

Cut-off values for defining cell activation as “positive” and “negative”for ALS patients were determined by comparison with values from 37normal ALS-negative, healthy donors. Results are expressed as themean±SD. Statistical analysis was performed by GraphPad Prism 4.0Software (San Diego, Calif., USA), which included two-tailed t-test fortwo groups' comparison, and One-Way ANOVA (Newman-Keuls test) foranalysis of differences between multiple groups. Correlationrelationship was analyzed using Pearson's rank correlation coefficient.For all analysis, a value of P<0.05 was considered significant.

Example 1 Cross-Section Study of Immune Activation in ALS PatientsCompared to Normal Subjects

A cross sectional study of immune activation was performed on blood from40 patients diagnosed with ALS as compared to 37 controls with initialstatistical analyses performed independent of drug treatment status. ALSblood cells showed abnormal levels of activation. Table 2 summarizes theresults of this study.

TABLE 2 Comparative analysis of serum antibodies and differentiationantigen expression in blood of ALS patients and normal controls P ValueALS patients Normal Controls (ALS vs. Parameter (n = 40) (n = 37^(A))Controls) CD4/CD8 ratio 2.84 ± 1.53 2.20 ± 0.98 0.0261 % CD4 47.42 ±8.03  37.99 ± 11.96 <0.0001 % CD4CD38 27.14 ± 11.50 31.36 ± 10.69 0.0799Med CD4CD38^(B) 12.67 ± 14.24 18.83 ± 17.00 0.0784 % CD8 20.45 ± 8.01 19.85 ± 7.05  0.6986 % CD8CD38 13.35 ± 8.09  12.03 ± 4.53  0.3620 MedCD8CD38^(B) 3.30 ± 6.32 2.68 ± 4.18 0.6003 % CD14 2.31 ± 0.99 3.25 ±1.41 0.0002 Mean CD14DR^(C) 847.79 ± 228.55 566.59 ± 130.43 <0.0001 CD14SSC 465.9 ± 155.5 388.49 ± 162.24 0.0198 % CD14CD16 42.44 ± 11.03 24.31± 15.70 <0.0001 Serum-IgG (mg/ml) 8.05 ± 5.72 11.26 ± 5.57  0.0038Serum-IgM (mg/ml) 2.31 ± 2.27 1.37 ± 1.14 0.0171 ^(A)n = 80 for controlsamples for serum-IgG and -IgM. ^(B)Median CD38 fluorescence expressedon CD4 and CD8 T-Cell. ^(C)Mean HLA-DR fluorescence expressed on CD14monocyte.

Patients with ALS had significantly higher proportional levels of theCD4 T lymphocyte subset as compared to controls (P<0.0001). By contrast,the CD8 T cell level was similar in both patients and controls. Theseproportional differences from control indicate a significant increase inthe ratio of CD4/CD8 cells in patients with ALS (P=0.0261). No evidenceof lymphocytic activation above normal in T cell subsets was observed inpatients with ALS.

Compared to controls, the absolute percent of CD14 cells within thetotal white blood cell count in ALS patient blood was significantlydecreased (P=0.0002). CD14+ monocytes from patients with ALS expressedsignificantly higher than normal levels of major histocompatibility(MHC) antigen class II (HLA-DR) (P<0.0001) (Table 2). Perivascularmacrophages normally constitutively express MHC Class II (HLA-DR), whichis upregulated in response to injury (Streit et al. 1989. Expression ofIa antigen on perivascular and microglial cells after sublethal andlethal motor neuron injury. Exp. Neurol. 105:115-126). Modulation ofHLA-DR on blood monocytes has been associated with a variety ofpathogenic states and blood measurements have been shown to haveclinical significance (Gascon et al. 2002. Increased HLA-DR expressionon peripheral blood monocytes in subsets of subjects with primary HIVinfection is associated with elevated CD4 T-cell apoptosis and CD4T-cell depletion. J. Acquir. Immune. Defic. Syndr. 30:146-153; Gu et al.2003. Time course of proinflammatory and anti-inflammatory responsesafter cardiac operation: monocyte HLA-DR expression. Ann. Thorac. Surg.76: 654-655; Melichar et al. 2003. Phenotype and antitumor activity ofascitic fluid monocytes in patients with ovarian carcinoma. Int. J.Gynecol. Cancer. 13:435-443). Almost half of the CD14 cells in ALS bloodhad characteristics of tissue macrophages, expressing significantlyhigher than normal levels of the CD16 antigen (P<0.0001).

The aberrant monocytic phenotype defined by higher reactivity for MHCantigen class II (HLA-DR) and CD16 markers, were associated withsignificant differences in CD14-associated SSC (measure of granularityand differentiation) between patients with ALS and normal controls.Compared with controls, monocytes from ALS patients had statisticallyincreased granularity (higher SSC values) (P=0.0198).

Finally, the overall status of humoral immunity was evaluated byquantitating levels of serum-IgG and -IgM in patients with ALS andcontrols (Table 2); serum-IgG levels in patients with ALS weresignificantly lower than controls (P=0.0038), whereas, serum-IgMconcentrations were significantly higher (P=0.0171).

Example 2 CD4 T-Cell Activation is Decreased in Advanced ALS Disease

To test whether T lymphocytic activation would be related to duration orseverity of disease, the T cell activation results were compared withthe clinical ALS values shown in Table 1. To simplify clinicalcorrelative analyses, patients were divided into two groups based on theALSFRS-R scale (0-48, no disease=48). Those with severe impairment (anALSFRS-R score of 0-24, n=10) were compared to those with milderimpairment (ALSFRS-R score >24, n=26).

As shown in FIG. 1, T cell activation levels as quantitated by detectionof CD38 antigens on the surface of CD4 T cells was significantlydifferent between the two groups (P<0.05). Compared with controls,CD4/CD38 reactivity was significantly lower in patients with ALSFRS-Rscore of 24 or lower (P<0.01) whereas no difference of CD4/CD38reactivity was found in ALS patients with less severe disease (ALSFRS-Rscore >24). No significant disease associated changes were observed inany of the other T cell (CD4 or CD8) parameters measured.

Example 3 Macrophage Activation and ALS Disease Progression

To evaluate whether systemic monocyte/macrophage activation would berelated to duration or severity of disease, macrophage activationparameters from Table 2 were plotted against clinical measures ofdisease severity to test whether any disease specific changes would bepresent.

Levels of CD14 cells (as a proportion of total white cell count) did notvary between individuals with mild or severe disease. There was asignificant correlation between the level of monocyte/macrophageactivation with severity of disease defined by ALSFRS-R score (Pearsonr=−0.3464, P=0.0409) (FIG. 2 a).

When the rate of ALS disease progression (ALSFRS-R score change permonth) was compared to CD14 cell HLA-DR expression, a direct andsignificant relationship was observed. FIG. 2 b shows that higherCD14-DR levels were associated with a more rapid progression of ALSdisease (Pearson r=0.3696, P=0.0265). Finally, the elevated level ofmacrophage differentiation antigen CD16 co-expression on the CD14expressing monocytes was independent of severity of disease.

Example 4 Changes of Serum-IgG and -IgM in Patients with ALS

Table 2 shows that the concentration of IgG and IgM in serum wassignificantly different in patients with ALS as compared to normalcontrols. Levels of serum-IgG and -IgM also varied with diseaseseverity. ALS patients with ALSFRS-R scores of 0-24 had significantlylower levels of serum-IgG than normal controls (P<0.05) and serum-IgGlevels were similar in both individuals with milder disease and controls(FIG. 3 a). However, serum-IgM levels were significantly higher inindividuals with milder disease (P<0.01) and not significantly differentbetween normal controls and in individuals with severe disease (FIG. 3b).

Example 5 Therapy Related Changes in ALS Specific Immune ActivationStatus

Table 1 shows the medications that patients with ALS were taking at thetime of assessment in the current study. The drugs fell into twodifferent categories; riluzole approved for slowing ALS diseaseprogression and nonsteroidal anti-inflammatory drugs (NSAIDS). Table 3summarizes the effects of medication treatments on immune activationmeasurements in patients with ALS. In particular, levels of macrophageactivation and differentiation as measured by HLA-DR and CD16 did notchange with therapy.

TABLE 3 Comparative analyses of serum antibodies and differentiationantigen expression in blood of normal controls and ALS patients with orwithout medications ALS patients riluzole + Untreated riluzole NSAIDSNormal Controls Parameter (n = 6) (n = 20) (n = 10) (n = 37)^(A) CD4/CD82.59 ± 1.46 2.77 ± 1.54 2.74 ± 1.44 2.20 ± 0.98 % CD4 44.62 ± 8.09 46.10 ± 7.45  49.02 ± 9.20  37.99 ± 11.96 % CD4CD38 20.52 ± 7.92  27.88± 10.76 30.81 ± 14.87 31.36 ± 10.69 Med 5.91 ± 5.47 12.76 ± 12.62 19.75± 20.12 18.83 ± 17.00 CD4CD38^(B) % CD8 21.25 ± 9.59  20.20 ± 7.69 21.43 ± 8.27  19.85 ± 7.05  % CD8CD38 15.05 ± 6.70  13.20 ± 9.62  12.47± 5.49  12.03 ± 4.53  Med 2.18 ± 1.99 4.90 ± 8.55 1.70 ± 2.16 2.68 ±4.18 CD8CD38^(B) % CD14 2.39 ± 1.10 2.28 ± 1.22 2.33 ± 0.66 3.25 ± 1.14Mean 779.95 ± 336.69 839.02 ± 220.79 829.43 ± 181.55 566.59 ± 130.43CD14DR^(C) CD14 SSC 509.6 ± 220.3 457.4 ± 153.2 467.2 ± 162.3 388.5 ±162.2 % CD14CD16 41.20 ± 8.19  44.22 ± 12.91 37.83 ± 8.62  24.31 ± 15.70Serum-IgG 8.82 ± 5.77 7.90 ± 4.58 8.59 ± 8.35 11.26 ± 5.57  (mg/ml)Serum-IgM 2.48 ± 1.08 2.78 ± 3.03 1.53 ± 0.87 1.37 ± 1.14 (mg/ml) ^(A)n= 80 for control samples for serum-IgG and -IgM. ^(B)Median CD38fluorescence expressed on CD4 and CD8 T-Cell. ^(C)Mean HLA-DRfluorescence expressed on CD14 monocyte.

Even the inclusion of NSAIDS was not associated with lower levels ofmacrophage activation (Table 3). Similarly, there were no significantdifferences between patients in the three treatment categories regardingthe levels of CD4/CD38 co-expression and serum-IgG. However dual therapy(riluzole+NSAIDS) was associated with normalization of serum-IgM levels,whereas, the riluzole alone group was no different from untreatedpatients.

Discussion Relating to Examples 1-5

In the current study, immunophenotypic analyses and humoral immunityassessment was performed on blood from patients with ALS to determinewhether systemic immune alteration might be present in ALS. Persistentlyactivated macrophages were observed in the blood of patients with ALS.The high levels of macrophage activation and differentiation werepersistent throughout the course of ALS. In addition, macrophageactivation defined by CD14 co-expression of HLA-DR became even higher ina disease severity related manner, and was directly related to the rateof disease progression. Moreover, the macrophage activation status wasnot improved in ALS patients treated by riluzole (the only currentlyapproved treatment for ALS) and NSAIDS. The direct relationship betweendegree of blood macrophage activation and rate of ALS diseaseprogression indicates a link between the blood and pathogenic processesongoing in the CNS.

The significantly higher levels of HLA-DR on the circulating monocytesin patients with ALS may be attributed to the reaction of peripheralimmune system to motor neuron injury, extending the reaction ofmicroglia/macrophages in the spinal cord and brain in patients with ALS.Alternatively, and as suggested by FIGS. 2 a-2 b and as observed in HADand SIVE, activated macrophages in the blood of patients with ALS maycommunicate with spinal cord perivascular areas and play a directpathogenic role in disease

The high levels of HLA-DR on ALS CD14 cells was coupled with anelevation in the proportion of CD14 cells co-expressing the tissuemacrophage marker, CD16 in ALS. CD14+/CD16+ monocytes are asubpopulation of cells that while in the circulation acquire features incommon with mature tissue macrophages. They are able to producepro-inflammatory cytokines, such as TNFalpha, IL-1alpha, and IL-6, buttheir expression of the potent antiinflammatory cytokine IL-10 is low orabsent. Therefore, CD14+/CD16+ cells may induce more pronounced levelsof inflammation than regular monocytes.

CD14+/CD16+ monocytes can rapidly migrate to the site of inflammation,where they readily mature into proinflammatory macrophages. Withoutbeing held to theory, neurological disorders such as Alzheimer's disease(AD) and AIDS-related dementia may be due in part to neurotoxic factorsreleased by these cells when migrating into the CNS and crossing theBBB. Elevated levels of HLA-DR expression on CD16 expressing monocytesmight result in blood monocytes migrating into the CNS and crossing theBBB in ALS, by mechanisms similar to the activated macrophages in AD andHAD. The decrease of the absolute percent of CD14 cells in patients withALS may be associated with the migration of circulating CD14/CD16+ cellsto perivascular regions of disease, where these cells release localneurotoxic factors such as IL-6, a factor implicated as potentiallyplaying pathogenic roles in ALS (Ono et al. 2001. Increasedinterleukin-6 of skin and serum in amyotrophic lateral sclerosis. J.Neurol. Sci. 187:27-34; Sekizawa et al. 1998. Cerebrospinal fluidinterleukin 6 in amyotrophic lateral sclerosis: immunological parameterand comparison with inflammatory and non-inflammatory central nervoussystem diseases. J. Neurol. Sci. 154:194-199), that could damage themotor neurons, similar to AIDS-related dementia and other HIV-associatedneurological disorders.

Although blood macrophage abnormalities persisted throughout thepathogenic ALS process in this cross sectional study, T-cellmeasurements showed changes related to disease severity. Compared withnormal controls, ALS patients had a significant increase in T cellsexpressing CD4, however, the percentage of CD8 T cells was found to bein the normal range, resulting in a significant increase in the ratio ofCD4/CD8 cells in ALS. The increased proportions of CD4+ T-cells and theincreased CD4/CD8 ratio in the peripheral blood of the ALS patientsdescribed herein suggests a possible shift of the immune balance eithertowards anergy or the Th2 type humoral response rather than a Th1 typecellular immune response. This Th2-like lymphocytic immune responsecould be induced by the presence of high levels of activated CD14+/CD16+monocytes in ALS. FcγR (CD16) ligation on activated macrophages maychange the phenotype of these activated macrophages to cells thatpreferentially drive a Th2-like response and result in the alteration ofthe Th1 type adaptive component of the immune system.

Concentrations of serum-IgG and -IgM antibodies were significantlydifferent compared to normal controls, and also changed with diseaseprogression. Patients with ALS had a normal IgG concentration and higherlevels of IgM in early stage of disease. Lower levels of serum-IgG witha concomitant normalization in serum-IgM secretion were observed withdisease progression in patients with ALS. Normalization of serum-IgM inALS patients was associated with combined therapy. The change of serumantibody levels in ALS patient blood might relate to persistentmacrophage activation driving CD4 T-cell dysfunction and/or defectiveTh1 type immunity.

In the study of T cell activation markers, CD38 levels decreased on CD4T cells with ALS disease progression. However, the CD8/CD38 reactivityremained within the normal range. These data suggest that the adaptivecomponent of the (T cell) immune system did not become active during ALSpathogenesis. Our observations on blood from patients with ALS suggeststhat lymphocytes, unlike microglia/macrophages, play a minor role in theactive ALS spinal cord associated immune-inflammatory reaction.Therefore, the neuroinflammatory process in ALS may be minimallydependent upon lymphocyte infiltration but rather is driven bymacrophage activity.

The inventors have for the first time demonstrated a systemic alterationof blood cell activation in patients with ALS. Persistentdisease-associated macrophage activation was observed in ALS blood andlevels of HLA-DR on CD14 cells was directly associated with rate of ALSdisease progression. The current study confirms systemic macrophageactivation in ALS disease, implicating an active role of macrophages inALS pathogenesis. Abnormally activated macrophages without evidence ofconcomitant T-cell activation was observed in ALS blood. Theseobservations indicate that systemic immune dysregulation plays a role inthe pathogenesis of ALS. The data presented here indicates that ALS maybe a kind of systemic inflammatory disease with local manifestationscausing motor neuron loss.

These observations are the basis for the methods of the invention formonitoring ALS disease progression, which can be accomplished bymeasuring the activation- and inflammation-related markers ofcirculating monocytes, such as HLA-DR and CD16, as well as the status ofT-cell activation in patients with ALS. The invention provides valuableassistance in monitoring the treatment of ALS as an immune dysfunctiondisease. Moreover, These observations are also the basis for theinvention as it relates to therapeutic intervention aimed at reducinginflammation in ALS.

Example 6 Treatment of Two ALS Patients with WF10

Two patients, diagnosed with ALS after 2001, received WF10 (also knownas IMMUNOKINE™). The drug in each case was used at the same dose withthe same interval between doses for each patient. The dose, 0.3 cc/kg,was given intravenously for days as a one hour infusion (0.5 cc/kg ofWF10, a 63 mM solution of chlorite containing solution, infused over 1hour in 500 cc of saline each day for five days). This regimen wasrepeated every three weeks. One cycle thus was composed of 5 days of 1hour infusions followed by 3 weeks without receiving drug. Patient 1received 5 cycles; patient 2 received 4 cycles. No adverse side effectswere noted in either patient.

Patient 1 is 59 y.o. woman with a familial form of ALS (a known mutationin the superoxide disniutase gene, SOD) who showed a progressive loss offunction as measured by the standard ALS functional rating score(ALS/FRS) from the time of diagnosis (score of 40) until the time ofinitiation of WF10 therapy 21 months later with a score of 15. In astandard ALS patient, the rate of ALS progression based on the ALS/FRSscoring system is essentially linear, with ALS progressing at apredictable rate after the slope of decline is known. In this case, aswell as in the second patient's case, the predicted rate of diseaseprogression is shown as a projected dotted line extending from the soliddeclination lines in the ALS/FRS scores. At the time of therapyinitiation the Patient 1 could no longer swallow food or fluids and hadhad a gastrointestinal tube (G-tube) placed into her stomach for feedingpurposes. The inability to eat is a sign of brain involvement with thedegenerative ALS process, whereas the ALS/FRS measurement documents thespinal cord degeneration.

After the first cycle of WF10, the patient had a dramatic improvement ofher symptoms, including: restoration of the ability to swallow and eat,leading to the removal of the G-tube (dotted line on the graph denotestime of G-tube placement, the removal is shown as a solid line and aftertherapy was discontinued, the dotted line shows the placement of a newG-tube), halting of facial fasciculations and vocal waivering (bothsymptoms of worsening neurologic disease that stopped). During the timeof therapy and through 2 months after discontinuation of therapy theALS/FRS score remained stable at for 7 months, at a time when she wouldhave been predicted to have progressed to a 0 within 5 months absenttherapy. She was able to eat by mouth for 8 months after therapyinitiation whereas she was never expected to eat after placement of theG-tube. Due to inability to obtain drug after the 5th cycle, shediscontinued therapy after 7 months of stable disease and within thenext 6 months her ALS disease progressed at a rate identical to her ratepretreatment. Based on the curves shown in FIG. 6, the patient showed abeneficial effect both in the ALS/FRS score of 7 months diseasestability and in the reversal of brain based symptoms (bulbar symptoms)of her inability to eat of 8 months. No currently approved or knownexperimental drug has ever reversed bulbar symptoms and no drug hascaused the ALS/FRS score to stabilize.

Patient 2: This 37 y.o. man, diagnosed with a sporadic (non familial)form of ALS in 2003, had an ALS/FRS score of 40 when diagnosed andwithin a year had progressed rapidly as shown in FIG. 6. At the time ofWF10 therapy he had just had a G-tube placed, as he could no longerswallow. Within a week of WF10 therapy his ability to eat was restoredand the G-tube was removed, similar to the clinical response inpatient 1. The patient received 4 cycles of WF10 during which hisALS/FRS score remained stable at 21, which allowed him to continuewalking with a walker and interacting with his family. His quality oflife improved dramatically with initiation of therapy. At the time ofpatent filing, he has continued to eat and his ALS/FRS score remained at21, both significant responses lasting for 5 months. As above, notherapy has shown this type of effect.

FIG. 4 is shows changes in blood macrophage activation results inPatient 1 as a result of treatment with WF10. The Y axis representsUnits of HLA-DR expressed on the surface of blood CD14 cells(monocyte/macrophages). The second column (ALS rapid) shows the level ofDR expression exhibited by ALS patients with a rapidly decliningclinical course. The third column (ALS slow) shows the level of DRexpression in an ALS patient with slowly progressive disease. Theprogression rates between these two columns differs by approximately5-10 fold (FIGS. 2 a and 2 b). The patients with high levels of DRprogress 5-10× faster than those with low levels of DR.

The first set of columns in FIG. 4 shows the baseline level of DR (high,fast progressor) in the ALS patient, with the second column representingthe level of DR expressed three weeks after one 5 day cycle of WF10 (0.5cc/kg of WF10, a 63 mM solution of chlorite containing solution, infusedover 1 hour in 500 cc of saline each day for three days). The thirdcolumn in the first set of columns represents the normal (38 normalblood donor composite) level of DR expression on CD14 cells+/−1 standarddeviation.

As shown in FIG. 4, the level of HLA-DR on the circulating bloodmonocytes (CD14+ cells) in Patient 1 was shown to shift from an elevatedlevel to a normal level after one cycle. In a recent paper (Zhang et al,J NeuroImmunology 2005 159:215-224) the level of DR on monocytes wassignificantly associated with ALS disease progression rate. The datashown in this figure compare the rates of a rapid progressor with a slowprogressor and show that Patient 1's blood monocytes converted from arapid to a slow phenotype with WF10 administration. This data inconjunction with the clinical data shown in FIG. 6 suggest that theregulation of systemic macrophage activation (also as shown in FIG. 5for MS) with a chlorite based drug can be monitored by both blood testsand clinical observation.

These data demonstrate that WF10 administration was associated withsymptomatic improvement in a rapidly progressing ALS patient at the sametime that blood values of macrophage activation resolved, e.g.,reduction in pathologic macrophages was concomitant with improvement inthe patient.

Example 7 Treatment of Ms Patient with WF10

FIG. 5 is a composite set of curves representing blood macrophageactivation measurements taken from a patient with multiple sclerosis(MS) who received WF10 therapy as described in Example 6 above with onecycle of WF10. The values along the Y axis represent the ratio of theobserved measurement for each of the parameter measured divided by thenormal level (38 normal donor mean value) to yield a ratio. Day 0represents baseline values for 5 different macrophageactivation/proliferation markers. Each of the 5 markers were elevatedbeyond normal range (shown by the solid and dotted lines) at Day 0.

The patient was then treated with one cycle of WF10 as above and twosubsequent blood studies were performed 14 and 28 days after initiationof the 3 day course of WF10. Macrophage proliferation (CD14Ki67,CD14PCNA) and activation (CD14/DR, CD14SSC, CD14/16%) all shiftedtowards the normal range on day 14 showing a response to one cycle ofWF10 in 5/5 macrophage parameters measured. Two weeks later (day 28) thevalues had essentially returned to pretreatment levels. These data areconsistent with a drug induced effect on abnormal macrophageproliferation/activation parameters in a patient with multiplesclerosis.

Example 8 Analysis of Macrophages of ALS and AD Patients

A cross-sectional study of immune activation was performed on blood from38 patients diagnosed with sALS as compared to control groups withinitial statistical analyses performed independent of drug treatmentstatus. In the present investigation two control groups were chosen tocompare with sALS patients: 28 age-matched normal controls and 25 ADpatients as neurological disease controls. Blood cells from patientswith sALS, similar to disease control AD patients, showed abnormallevels of activation. Table 4 summarizes the results of this study.Patients with sALS and AD had significantly higher proportional levelsof the CD4 T lymphocyte subset as compared to normal controls (p<0.05).By contrast, the CD8 T-cell level and the ratio of CD4/CD8 were similarin all three groups. No evidence of lymphocytic activation above normalin T-cell subsets was observed in patients with sALS and diseasecontrols.

TABLE 4 Comparative analysis of serum antibodies and differentiationantigen expression in blood of sALS patients, normal controls and ADNormal P VALUE P VALUE P Value sALS Controls AD (SALS VS. (AD VS. (sALSParameter (n = 38) (n = 28) (n = 25) NORMAL) NORMAL) vs. AD) CD4/CD82.87 ± 1.56 2.33 ± 1.59 3.43 ± 2.72 NS NS NS % CD4 47.43 ± 8.04  39.81 ±11.30 47.37 ± 11.22 <0.01  <0.05  NS % CD4CD38 27.21 ± 11.76 32.24 ±10.51 25.67 ± 12.02 NS NS NS Med CD4CD38 ^(a) 13.02 ± 14.54 19.15 ±16.71 14.08 ± 16.12 NS NS NS % CD8 20.38 ± 8.22  21.19 ± 8.53  19.93 ±12.43 NS NS NS % CD8CD38 13.67 ± 8.20  12.41 ± 6.85  15.86 ± 11.46 NS NSNS Med CD8CD38 ^(a) 3.43 ± 6.46 1.93 ± 2.40  5.85 ± 16.19 NS NS NS %CD14 2.34 ± 1.01 2.51 ± 0.93 2.49 ± 1.00 NS NS NS Mean CD14DR ^(b)825.60 ± 206.62 582.56 ± 144.35 911.93 ± 341.80 <0.001 <0.001 NS CD14SSC ^(c) 466.3 ± 159.6 346.5 ± 42.3  434.7 ± 226.5 <0.01  NS NS %CD14CD16 42.44 ± 11.22 23.90 ± 10.60 41.77 ± 18.97 <0.001 <0.001 NSSerum-IgG (mg/ml) ^(d) 7.80 ± 5.76 11.26 ± 5.57  ND ^(e) <0.003 ND NDSerum-IgM (mg/ml) ^(d) 2.28 ± 2.30 1.37 ± 1.14 ND   <0.03  ND ND ^(a)Median CD38 fluorescence expressed on CD4 and CD8 T-Cell. ^(b) Mean DRfluorescence expressed on CD14 monocyte. ^(c) CD14-associated sidelight-scatter characteristics. ^(d) n = 80 for control samples forserum-IgG and -IgM. ^(e) ND, not data

Analysis of monocyte/macrophage markers showed that CD14+ monocytes frompatients with sALS and AD expressed significantly higher than normallevels of major histocompatibility (MHC) antigen class II (HLA-DR)(p<0.001) but no difference was found in the absolute percent of CD14cells within the total white blood cell count in either of the sALS andAD patient blood specimens as compared to normal controls (Table 4).Almost half of the CD14 cells in sALS and AD blood had characteristicsof tissue macrophages, expressing significantly higher than normallevels of the CD16 antigen (p<0.001). The aberrant monocytic phenotypedefined by higher expression of HLA-DR and CD16 was associated withsignificant differences in CD14-associated SSC (measure of granularityand differentiation) between patients with sALS and normal controls.Compared with normal controls, monocytes from sALS patients hadstatistically increased granularity (higher SSC values) (pb0.01).Finally, the overall status of humoral immunity was evaluated byquantitating levels of serum-IgG and -IgM in patients with sALS andnormal controls (Table 4); serum-IgG levels in patients with sALS weresignificantly lower than normal controls (p<0.003), whereas, serum-IgMconcentrations were significantly higher (p<0.03) (sera from the ADpatients were not available for study).

The high levels of macrophage activation and differentiation werepersistent throughout the course of sALS. In addition, macrophageactivation defined by CD14 co-expression of HLA-DR was directly relatedto the rate of sALS disease progression. Moreover, the macrophageactivation status was not improved in sALS patients treated by riluzole(the only currently approved treatment for ALS) or NSAID. The directrelationship between degree of blood macrophage activation and rate ofALS disease progression suggests a link between the blood and pathogenicprocesses ongoing in the CNS.

The preceding merely illustrates the principles of the invention. Itwill be appreciated that those skilled in the art will be able to devisevarious arrangements which, although not explicitly described or shownherein, embody the principles of the invention and are included withinits spirit and scope. Furthermore, all examples and conditional languagerecited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1.-20. (canceled)
 21. A method comprising: assessing a level ofactivated monocytes and/or proliferating macrophages in a biologicalsample obtained from an individual, wherein said individual has or issuspected of having a macrophage-associated neurological disorder,wherein said individual is receiving a treatment, and wherein saidtreatment comprises administration of chlorite; wherein said assessingprovides guidance for providing or altering a level of said treatment.22. The method of claim 21, wherein said macrophage-associatedneurological disorder is amyotrophic lateral sclerosis (ALS).
 23. Themethod of claim 21, wherein said assessing comprises assessing a levelof activated monocytes by measuring one or more markers that indicatemonocyte activation, differentiation and/or proliferation.
 24. Themethod of claim 21, wherein said assessing comprises assessing a levelof activated monocytes by measuring a level of HLA-DR in a population ofCD14+ cells and/or the presence of CD16+ cells in a population of CD14+cells and/or the presence of CD14+/CD16+ cells in said sample.
 25. Themethod of claim 21, wherein said assessing comprises assessing a levelof proliferating macrophages by measuring an elevation in proliferationand/or activation markers relative to non-disease blood macrophages. 26.The method of claim 21, further comprising comparing said level ofproliferating macrophages to a normal level of proliferatingmacrophages.
 27. The method of claim 21, wherein said chlorite isadministered in a form of a matrix of chlorite ions.
 28. The method ofclaim 21, wherein said chlorite is administered in an aqueousformulation.
 29. The method of claim 21, wherein said chlorite isadministered in a form of a pharmaceutically acceptable chlorite salt.30. The method of claim 29, wherein said chlorite salt is sodiumchlorite.
 31. A method of monitoring therapy in a subject receivingtreatment for ALS, the method comprising: assessing a level ofpathologic macrophages in a sample obtained from a patient receivingtreatment for amyotrophic lateral sclerosis (ALS), wherein the treatmentcomprises administration of chlorite, and comparing the level ofpathologic macrophages to a normal level of pathologic macrophages. 32.The method of claim 31, wherein said assessing said level of pathologicmacrophages comprises measuring one or more markers that indicatemonocyte activation, differentiation and/or proliferation.
 33. Themethod of claim 31, wherein said assessing said level of pathologicmacrophages comprises measuring a level of HLA-DR in a population ofCD14+ cells and/or the presence of CD16+ cells in a population of CD14+cells and/or the presence of CD14+/CD16+ cells in said sample.
 34. Themethod of claim 31, wherein said proliferating macrophages exhibit anelevation in proliferation and/or activation markers relative tonon-disease blood macrophages.
 35. The method of claim 31, wherein saidchlorite is administered in a form of a matrix of chlorite ions.
 36. Themethod of claim 31, wherein said chlorite is administered in an aqueousformulation.
 37. The method of claim 31, wherein said chlorite isadministered in a form of a pharmaceutically acceptable chlorite salt.38. The method of claim 37, wherein said chlorite salt is sodiumchlorite.